Atherosclerotic coronary artery disease is a major cause of morbidity and mortality in western nations. A number of risk factors have been identified that can dramatically increase the risk of developing atherosclerotic disease. One of these factors is high serum cholesterol levels. Fat and cholesterol deposit on blood vessel walls, straining the blood flow. This eventually causes injury at the site and leads to the formation of damaging blood clots. When these blood clots reach the heart, they are life threatening.
Another risk factor for atherosclerotic disease, identified in a large number of studies, is elevated blood levels of plasminogen activator inhibitor type 1 (PAI-1). For example, PAI-1 levels are elevated in young survivors of myocardial infarction (Hamsten, 1985) and a diurnal variation in PAI-1 levels corresponds to the diurnal pattern of myocardial infarction (Krishnamurti et al., 1992). In addition, PAI-1 mRNA levels are elevated in atherosclerotic arteries (Schneiderman, 1992), and PAI-1 levels in plasma are positively correlated with the risk of recurrent myocardial infarction (Hamsten, et al., 1987).
Normal hemostasis in humans depends on the delicate balance between coagulation and fibrinolysis. Accordingly, any imbalance in this system either towards a hypercoagulable condition or a hypofibrinolytic state may result in the development of thrombotic vascular disease. Current interventions for pathological thrombosis rely primarily on thrombolytic therapies or on direct physical ablation of the offending thrombus, by angioplasty or other surgical techniques. These regimens are then generally followed by administration of potent anticoagulants in order to maintain vessel patency. While such treatments have a high degree of effectiveness in removing the original blockage they do carry a significant risk of bleeding complications. Furthermore, approximately 40% of patients receiving thrombolytic therapy do not attain optimal reperfusion (Lincoff et al., 1993). In addition, 25% to 55% of patients undergoing angioplasty suffer from significant restenosis within six months (Cercek et al., 1991). Therefore, the development of additional treatment strategies, directed at enhancing fibrinolysis, may be effective in patients that do not currently respond to thrombolytics. Such a adjuvant therapy might also permit a reduction in anticoagulant therapy in all patients, lessening the potential risk of bleeding complications.
In vivo, natural thrombolysis is an orderly process that develops temporally through activation of the fibrinolytic system. The common end point of this pathway is conversion of the zymogen plasminogen into the trypsin-like protease plasmin, a proteolytic enzyme which through a series of specific cleavages systematically degrades fibrin into soluble fibrin degradation products, resulting in timely blood clot dissolution. The primary activators of plasminogen in vivo are the serine proteases, urokinase (uPA) and tissue plasminogen activator (tPA), with the latter being the major physiologic activator of plasminogen in plasma (Wallen, 1980; Saksela, 1988). In addition to the regulation of vascular fibrinolysis (Bachmann et al., 1987), these enzymes are also thought to critically influence many other biological processes including ovulation (Hsueh et al., 1988; Ohlsson et al., 1991), inflammation (Pollanen et al., 1991), tumor metastasis (Dano et al., 1985), angiogenesis (Moscatelli et al., 1988), and tissue remodeling (Saksela, 1988), and smooth muscle cell proliferation (Grainger et al., 1993).
The regulation of fibrinolysis is a complex process that is controlled on many levels. The synthesis and release of PAs is governed by various hormones, growth factors, and cytokines (Saksela, 1988; Dano et al., 1985). Following secretion, PA activity can be regulated both positively and negatively by a number of specific protein:protein interactions. Activity can be enhanced or concentrated by interactions with fibrin (Hoylaerts et al., 1982), the uPA receptor (Ellis et al., 1991), the tPA receptor (Hajjar et al., 1990) or the plasminogen receptor (Plow et al., 1991). In contrast, PA activity can be down-regulated by the presence of specific PA-inhibitors (PAIs) Kruithof, 1988; Hart, 1988) or by direct plasmin inhibition (Aoki, 1989). The overall activity of the fibrinolytic system is determined by the interactions among these various elements, and the balance between the opposing activities of enzymes and inhibitors.
Inhibition of thrombin is critical during and after thrombolytic therapy in order to prevent additional fibrin formation and platelet activation at the site of plaque rupture. Heparin, a cofactor for antithrombin III, is the primary agent used to inhibit thrombin in patients with acute coronary syndromes. For t-PA, the co-administration of intravenous heparin appears to be of particular importance in maintaining vessel patency, as evidenced by three recent randomized trials (Lincoff et al., 1993). Conversely, the thrombolytic efficacy of streptokinase is not significantly affected by heparin (GISSI 2 Investigators, 1990). This difference between t-PA and streptokinase is probably explained by several factors, including the shorter half-life of t-PA, its fibrin specificity, and the systemic fibrin(ogen)olytic state generated by streptokinase.
Coronary patency rates of as high as 85% at 90 minutes have been achieved with recent accelerated dosing regimens of t-PA, suggesting that there exists little room for improvement in thrombolytic therapy. However, this seemingly excellent rate of repeffusion belies several limitations that greatly hinder the effectiveness of current thrombolytic strategies (Lincoff et al., 1993). First, patency at 60 minutes, instead of 90 minutes, is the optimal goal in acute MI, since reductions in mortality are as great as 50% in patients treated during the first hour of infarction, but tail-off rapidly thereafter. Secondly, recent studies suggest that only those individuals who attain complete reperfusion--i.e., the restoration of rapid (TIMI grade 3) coronary flow--derive significant reductions in infarct size and mortality from lytic therapy. Yet, only 57% of patients attain TIMI 3 flow after receiving lytic agents. And finally, the significant incidence of reocclusion following initially successful thrombolysis (10-15%) results in a further reduction in the estimated number of patients who receive "optimal" thrombolysis, which has been estimated by Lincoff and Topol to be as low as 25% of patients (Lincoff et al., 1993). Hence, new strategies that result in faster, more complete, and sustained reperfusion are necessary.
It is not known why some patients attain rapid reperfusion following the initiation of thrombolytic therapy, whereas other patients exhibit delayed or incomplete reperfusion, and others no reperfusion. These clinical observations suggest the presence of an intrinsic resistance to thrombolysis that can vary substantially between patients. The mechanisms underlying thrombolysis resistance are poorly understood, and probably differ significantly depending on which plasminogen activator is administered. In the case of t-PA, local factors mediated by platelet activation appear to play a dominant role in determining thrombolytic efficacy. In the case of streptokinase, thrombolysis resistance may be a systemic phenomenon--i.e., mediated by anti-streptokinase antibodies that can inactivate streptokinase before it reaches the target thrombus.
Histologic and angioscopic examination of coronary thrombi reveals that they are frequently platelet-rich (Warnes et al., 1984). Jang et al. have demonstrated that platelet-rich arterial thrombi in rabbits are much more resistant to lysis by t-PA than erythrocyte-rich thrombi, even when t-PA is administered in pharmacologic concentrations (Jang et al., 1989). Among several possibilities, release of platelet plasminogen activator inhibitor-1 (PAI-1) and platelet-mediated clot retraction appear to be the dominant mechanisms underlying platelet-mediated clot lysis resistance in vitro (Levi et al., 1992, Kunitada et al., 1992).
PAI-1, a member of the serpin superfamily of protease inhibitors, is a fast-acting and specific inhibitor of t-PA (Loskutoff, 1989). PAI-1 is secreted by endothelial cells, and is normally present in plasma in trace amounts (Loskutoff, 1989). Human platelets contain approximately 4000 molecules of PAI-1 per cell (Sprengers et al., 1986), the majority of which exists in an inactive, or latent, form (Booth et al., 1988). Latent PAI-1 can be activated in vitro, and possibly in vivo (Vaughan et al., 1990). The sequence of PAI-1 including the leader sequence is disclosed in SEQ ID NO:7.
Monoclonal antibodies to PAI-1 markedly diminish the capacity of platelets to inhibit t-PA-mediated fibrinolysis in vitro, suggesting that sufficient active PAI-1 is present in platelet-rich clots to inhibit t-PA, and that PAI-1 is the dominant factor underlying platelet-mediated clot lysis resistance (Levi et al., 1992; Braaten et al., 1993). However, Kunitada et al. found no significant PAI-1 effect in a clot lysis system in which platelets inhibited thrombolysis initiated by pharmacologic concentrations of t-PA (Kunitada et al., 1992). These authors concluded that platelets inhibit clot lysis not via PAI-1, but rather as a consequence of clot retraction.
These discordant results may be explained by the different experimental conditions employed in these studies, since the magnitude of a PAI-1 effect observed in vitro is critically dependent upon the concentrations of t-PA and platelets. These concentrations may differ substantially between different experimental systems, and also may differ markedly from the concentrations of PAI-1, platelets, and t-PA that are attained at sites of clot formation in vivo. Whereas in vitro clot lysis assays typically employ a platelet concentration of 10.sup.8 -10.sup.9 /mL (Levi et al., 1992), histologic evaluation of platelet-rich coronary thrombi reveals that they consist, to a substantial degree, of solid masses of platelets (Warnes, 1984). Within this environment, the calculated concentration of platelets exceeds 10.sup.11 /mL. Hence, a more definitive assessment of the impact of PAI-1 vs. clot retraction on the lysis of platelet-rich clots requires the analysis of thrombi formed in vivo under conditions that mimic pathologic states, such as myocardial infarction. However, such studies have not been performed.
As previously discussed, the thrombolytic efficiency of t-PA is enhanced by co-administration of heparin (Lincoff et al., 1993). A resistance to prolongation of the activated partial thromboplastin time (APTT) is observed in some patients with acute ischemic syndromes, which may indirectly impede thrombolytic therapy (Maraganore et al., 1992; Hsia et al., 1992). However, the etiology, prevalence, and magnitude of systemic heparin resistance in the setting of acute coronary artery disease are unknown.
Localized heparin resistance at sims of arterial injury also occurs, and may be of greater clinical significance than systemic heparin resistance. Localized heparin resistance has been attributed to the capacity of thrombin to bind fibrin, since in vitro studies indicate that the heparin/antithrombin III complex is a poor inhibitor of clot-bound thrombin, compared to hirudin, a direct-acting thrombin inhibitor (Weitz et al., 1990). However, platelets contain several factors that inhibit heparin, such as heparatinase, platelet factor 4, and histidine-rich glycoprotein (Rucinski et al., 1983).
The accumulation of these factors within platelet-rich thrombi may also locally inhibit heparin efficacy, particularly under states of impaired coronary flow. To data, the relative impact of fibrin vs. platelet factors on heparin function in vivo has not been defined. Nevertheless, it is clear that platelet-rich thrombus formation is a thrombin-dependent phenomenon, since inhibition of thrombin formation at sites of arterial injury substantially reduces platelet deposition (Sitko et al., 1992).
In contrast to t-PA, successful thrombolysis by streptokinase (SK) is strongly associated with the attainment of a systemic lytic state, which generally is defined by a significant (e.g.&gt;20%) reduction in plasma fibrinogen and plasminogen (Rothbard et al., 1985). However, a significant portion of patients do not achieve a lytic state after receiving SK.
Streptokinase is highly immunogenic, and pre-existing anti-SK antibodies due to prior Streptococcal infection could prevent induction of a lytic state. Two studies as well as several case reports suggest that naturally occurring anti-SK antibodies can cause thrombolysis resistance (Lew et al., 1984; Hoffmann et al., 1988; Brugemann et al., 1993). However, another study indicates that pre-existing anti-SK IgG titers are not related to successful coronary thrombolysis (Fears et al., 1992), and the correlation between anti-SK IgG titers and streptokinase resistance in vitro is poor (Moran et al., 1984).
It is likely that several factors have prevented an adequate assessment of the anti-therapeutic impact of SK antibodies. First, the assays used in prior studies are relatively crude. For example, an anti-IgG titer does not discern between inhibitory vs. non-inhibitory antibodies. Similarly, the assay most commonly used for defining SK-resistance (i.e., identification of the maximal SK dilution factor that results in whole blood clot lysis within 10 min) is not independent of other inhibitors of fibrinolysis, such as .alpha.2-antiplasmin and PAI-1, which can vary substantially between patients. And finally, the clinical variable with which antibody titers are usually correlated (i.e., angiographically-documented arterial patency) suffers from several limitations. For example, this "snapshot" definition of successful thrombolysis (usually obtained 90-180 min after initiating therapy), does not differentiate between two patients who reperfused at 30 min and 80 min, respectively; yet their clinical benefit would probably differ significantly.
It is likely that a more refined analysis of the human immune response to streptokinase will yield information with important therapeutic implications. For example, clearance of SK from the circulation is probably highly dependent upon antibody-antigen interactions. Yet, very little is known about which regions of the SK molecule represent the dominant epitopes for antibody production in humans (Reed et al., 1993). Such information is also highly relevant to the readministration of SK to the significant number of patients who sustain a second myocardial infarction. Several studies suggest that anti-SK Ab titers sufficient to neutralize most or all of a standard SK dose (1.5 million units) can persist for as long as 1 year after receiving SK (Jalihal et al., 1990). Consequently, readministration of SK is not recommended for at least 1 year after receiving this agent. An identification of dominant SK epitopes might also allow the design of recombinant SK molecules with reduced immunogenicity (Marder, 1993), which could have considerable clinical impact.
During the last ten years the recognition of PAIs as critical regulators of the fibrinolytic system has gained broad acceptance. PAI-1, formerly called the endothelial cell PAI, or the fast acting plasma PAI, is thought to be one of the principal regulators of vascular fibrinolysis. It is a single chain glycoprotein with a molecular weight of 50 kDa (van Mourik et al., 1984) and is the most efficient inhibitor known of the single- and two-chain forms of tPA and of uPA, with second order rate constants ranging between 0.5 and 2.7.times.10.sup.7 M.sup.-1 s.sup.-1 (Lawrence et al., 1989).
PAI-1 is present in platelets and many other tissues and is produced by many cells in culture (Erickson et al., 1984; Sawdey et al., 1991; Krishnamurti et al., 1992). In vivo the primary extravascular source of PAI-1 appears to be vascular smooth muscle cells (Loskutoff, 1991). However, in response to endotoxemia or other pathological conditions, endothelial cells become a major site of PAI-1 synthesis (Pyke et al., 1991; Schneiderman et al., 1992; Keeton et al., 1993). In plasma PAI-1 is present as a complex with vitronectin or S protein (Wiman et al., 1984; Declerck et al., 1988; Wiman et al., 1988). PAI-1 is also associated with vitronectin in the extracellular matrix in culture, and may be involved in maintaining the integrity of the cell substratum in vivo (Mimuro et al., 1987; Knudsen et al., 1987; Mimuro et al., 1989). PAI-1 also binds to fibrin, but with a lower affinity (Braaten et al., 1993; Wagner et al., 1989; Keijer et al., 1991; Reilly et al., 1991, Reilly et al., 1992). The major source of plasma PAI-1 is not known but is likely to be the vasctilar smooth muscle cells. However, a contribution from the platelet pool cannot be excluded. PAI-1 functions efficiently in solution as well as when bound to surfaces and it is likely that it regulates fibrinolysis in both environments.
The cDNA for human PAI-1 was isolated in 1986. The single PAI-1 gene is composed of 9 exons spanning approximately 12 kb (Follo et al., 1989; Strandberg et al., 1988) and is a member of the serine protease inhibitor (serpin) super gene family (Huber et al., 1989; Carrell et al., 1986). Serpins are thought to inhibit their target proteases via a common mechanism that results in the generation of an equimolar, sodium dodecyl sulfate (SDS)-stable complex (Carrell et al., 1986). Though individual inhibitors generally demonstrate remarkable protease specificity, overall the reported serpin structures are strikingly similar (Huber et al., 1989; Stein et al., 1990; Loebermann et al., 1984; Mottonen et al., 1992). In native serpins the reactive center loop appears to be exposed on the surface of the molecule in a position where it can interact with its target protease (Schreuder et al., 1994; Carrell et al., 1994). A model of serpin function suggests that active serpins have mobile reactive center loops that can partially insert into .beta.-sheet A (Carrell et al., 1991; Carrell et al., 1992). Partial insertion results in the observed thermal instability of serpins but is necessary for function. Further insertion yields a latent inhibitor that is no longer reactive with the protease but has an increased thermal stability (Mottonen et al., 1992). Evidence for this model has come from studies where synthetic peptides, homologous to the reactive center loops of .alpha.1AT and antithrombin III, when added in trans, incorporate into their respective molecules, presumably as a central strand of .beta.-sheet A. This leads to an increase in thermal stability similar to that observed following cleavage of a serpin at its reactive center (Carrell et al., 1991; Schulze et al., 1990). The structural change also converts the serpin from an inhibitor to a substrate for its target protease (Bjork et al., 1992; Bjork, 1992; Schulze et al., 1991).
While most serpins are able to adopt different conformations, PAI-1 appears uniquely labile and in vivo exist in at least two distinct conformations, active and latent (Lawrence, 1989; Loskutoff et al., 1989). Active PAI-1 decays to the latent form with a half-life of approximately 1 hour at 37.degree. C. With exposure to denaturants (guanidine HCl or SDS), latent PAI-1 can be returned partially to the active form. Though recent X-ray crystallographic findings suggest a structural basis for these two conformations (Mottonen et al., 1992; Carrell et al., 1991), their biologic significance remains unknown.
Vitronectin, an adhesive glycoprotein present in plasma, platelets, and the extracellular matrix, binds active PAI-1 and appears to stabilize it in the active conformation (Declerck et al., 1988). The reaction of PAI-1/vitronectin complexes with either tPA or urokinase results in the dissociation of PAI-1 from vitronectin and the formation of PAI-1-PA complex. Negatively-charged phospholipids can convert latent PAI-1 to the active form, suggesting that cell surfaces may modulate PAI-1 activity (Lambers et al., 1987). The observation that latent PAI-1 infused into rabbits is apparently converted to the active form is consistent with this hypothesis (Vaughan et al., 1990). Kinetic and other evidence has also been presented for a second site of interaction between PAI-1 and tPA, outside of the PAI-1 reactive center (Chmielewska et al., 1988; Lawrence, 1990; Hekman, 1988). The precise localization for these various binding interactions remain unknown. Marked sensitivity of PAI-1 to inactivation by oxidants has been demonstrated, apparently involving a conformational change in PAI-1 upon oxidation of a critical Met residue (Strandberg et al., 1991; Lawrence et al., 1986). A similar sensitivity to oxidation has also been observed for other protease inhibitors and may represent a common mechanism for regulation of serpin activity in vivo, both in health and disease.
Vitronectin is a major protein component of plasma and is also found in many tissues. Synthesis is predominant in the liver, though platelets and monocytes also contain detectable protein. Deficiency of vitronectin has not been reported. Though vitronectin appears to serve an important function in cell adhesion, regulation of complement activation, and thrombosis, its precise function in vivo remains unknown (Tomasini et al., 1991).
The interaction of PAI-1 with vitronectin has generated considerable debate. In one study only active PAI-1 was shown to bind vitronectin (Sigurdardottir et al., 1990). However in another study no apparent difference in the binding was seen between active and latent PAI-1 (Kost et al., 1992). The reported dissociation constant is also controversial, with one group reporting a Kd of 0.3 nM for active PAI-1 and vitronectin (Seiffert et al., 1991), while another reports a major dissociation constant of 55-190 nM with a second, low capacity but high affinity binding site (Kd&lt;0.1 nM) (Salonen et al., 1989). Additional controversy surrounds the vitronectin binding site for PAI-1. One report, utilizing ligand blotting of vitronectin cyanogen bromide fragments, localizes the binding site to the somatomedin B domain at the N-terminus of vitronectin (Seiffert et al., 1991). In contrast a second study, using monoclonal antibodies, localizes the PAI-1 binding site to the C-terminus of vitronectin, between residues 348 and 370 (Kost et al., 1992). Some of these conflicting results may be explained by the interaction of both the active and latent forms of PAI-1 with vitronectin, but with markedly different affinities, along with differences in the relative composition of PAI-1 conformers present in alternative PAI-1 preparations. The vitronectin binding domain within PAI-1 is localized to a region near the N-terminus and includes portions of 60-helices C and E and .beta.-strand 1A (Lawrence et al., 1994).
In addition to stabilizing active PAI-1, vitronectin has been shown to alter its specificity, converting it to an efficient inhibitor of thrombin (Ehrlich et al., 1990; Keijer et al., 1991). Vitronectin-bound PAI-1 has a 200-fold greater second order rate constant toward thrombin than does free PAI-1. However this increase depends upon the source of vitronectin used. While all forms of vitronectin appear to bind PAI-1, only vitronectin isolated under physiological conditions is able to stimulate PAI-1 inhibition of thrombin (Naski et al., 1993). Vitronectin has also been shown to stimulate the inhibition of tPA by PAI-1, but to a much less dramatic extent (Keijer et al., 1991; Edelberg et al., 1991). In other studies, reactive center mutants of PAI-1 that have greatly reduced activity toward tPA were shown to have their activity partially restored in the presence of vitronectin (Keijer et al., 1991).
As noted, PAI-1 is thought to play a major regulatory role in a variety of biologic processes (Vassalli et al., 1991). Overexpression of PAI-1 may confer increased risk for thromboembolic disease and elevated PAI-1 levels have been associated with premature myocardial infarction (Krishnamurti et al., 1992; Hamsten et al., 1985). Increased levels of PAI-1 gene expression have recently been observed in atherosclerotic human arteries, suggesting a role for PAI-1 in the pathogenesis of atherosclerosis (Schneiderman et al., 1992).
PAI-1 may also play a major role in the clinical response to thrombolytic therapy with tPA. A number of investigators have identified two classes of thrombi, distinguished by the relative presence or absence of large numbers of platelets. Plateletrich thrombi have been observed to be particularly resistant to lysis by therapeutic thrombolytic agents in several animal models, as well as in humans (Krishnamurti et al., 1992; Jang et al., 1989). Recently, Levi, et al. (Levi et al., 1992) reported complete neutralization of platelet-dependent thrombolysis resistance in a rabbit model, using an anti-PAI-1 monoclonal antibody. In addition, Fay, (Fay et al., 1994) demonstrated that platelet rich clots deficient in PAI-1 were significantly more susceptible to tPA induced lysis than clots containing normal platelets, suggesting that PAI-1 is the major factor responsible for the resistance of platelet-rich thrombi to lysis, and lending further support to the notion that inhibition of PAI-1 activity might be a useful strategy for increasing the efficacy of thrombolytic therapy. Furthermore, inhibition of PAI-1 most likely is a safer form of treatment than administration of exogenous tPA.
As discussed, currently a very common treatment for thrombosis is the administration of tPA. While administration of tPA is effective in the short-term, for example in the treatment of acute coronary syndromes such as myocardial infarction, thrombolytic therapy is unsuccessful in approximately 20% of patients. Furthermore, coronary reocclusion following thrombolytic therapy is not uncommon. In many of these cases, tPA treatment is frustrated by the interference of endogenous PAI-1. This is especially true where there are a high level of PAI-1 rich platelets in the blood clots.
Another problem with therapy with tPA or other lytic agents such as urokinase plasminogen activator (uPA) or streptokinase, is that such agents can be administered only for short periods of time. That is because more prolonged therapy is associated with major hemorrhagic complications. Clearly, a safer and more effective treatment for thrombosis and cardiovascular disease is needed.
Fibrinolysis can be enhanced not only by administering exogenous clot dissolving agents such as tPA but also by inactivating PAI-1. In addition, thromboembolytic therapy with exogenous tPA, uPA, or streptokinase can be enhanced if supplemented with a synthetic PAI-1 inhibitor. Furthermore, the administration of a PAI-1 inhibitor to patients, is safer than the administration of excess lytic agents. Patients who have been identified with a complete deficiency of PAI-1 suffer from only a mild to moderate bleeding disorder (Dieval et al., 1991; Schleef et al., 1989; Lee et al., 1993, Fay et al., 1992). Thus, mild partial deficiency, induced by a PAI-1 inhibitory drug most likely represents a very safe, long-term, chronic treatment which may be of major benefit by reducing cardiovascular risk.